Eps15 Homology Domain Protein 4 (EHD4) is required for Eps15 Homology Domain Protein 1 (EHD1)-mediated endosomal recruitment and fission.

Upon internalization, receptors are trafficked to sorting endosomes (SE) where they undergo sorting and are then packaged into budding vesicles that undergo fission and transport within the cell. Eps15 Homology Domain Protein 1 (EHD1), the best-characterized member of the Eps15 Homology Domain Protein (EHD) family, has been implicated in catalyzing the fission process that releases endosome-derived vesicles for recycling to the plasma membrane. Indeed, recent studies suggest that upon receptor-mediated internalization, EHD1 is recruited from the cytoplasm to endosomal membranes where it catalyzes vesicular fission. However, the mechanism by which this recruitment occurs remains unknown. Herein, we demonstrate that the EHD1 paralog, EHD4, is required for the recruitment of EHD1 to SE. We show that EHD4 preferentially dimerizes with EHD1, and knock-down of EHD4 expression by siRNA, shRNA or by CRISPR/Cas9 gene-editing leads to impaired EHD1 SE-recruitment and enlarged SE. Moreover, we demonstrate that at least 3 different asparagine-proline-phenylalanine (NPF) motif-containing EHD binding partners, Rabenosyn-5, Syndapin2 and MICAL-L1, are required for the recruitment of EHD1 to SE. Indeed, knock-down of any of these SE-localized EHD interaction partners leads to enlarged SE, presumably due to impaired endosomal fission. Overall, we identify a novel mechanistic role for EHD4 in recruitment of EHD1 to SE, thus positioning EHD4 as an essential component of the EHD1-fission machinery at SE.

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